Module 2: Expression Engineering
20.109
Lecture 3
October 18th, 2007
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
STEM Education
best way to achieve Adequate Yearly Progress?
Content removed due to copyright restrictions.
Excerpt from Medina, Jennifer. “Making Cash a Prize for High Scores on Advanced Placement Tests.”
The New York Times, October 15, 2007.
[http://www.nytimes.com/2007/10/15/nyregion/15rewards.html, accessed May 8, 2008.]
Excerpt from Kula, Adam and Alexandra Frean. “Make science easier, examiners are told.” The Times,
August 29, 2007. [http://www.timesonline.co.uk/tol/news/uk/education/article2344054.ece, accessed
May 8, 2008.]
Excerpt from Cavanaugh, Sean. "Federal Rule Yields Hope for Science." Education Week, published online
October 5, 2007.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Expression Engineering Experiment
Day 1
Day 2
Day 3
Image of glowing luciferase
in a microcentrifuge tube,
removed due to copyright
restrictions.
RT
Figure by MIT OpenCourseWare.
Day 6
Day 5
Day 4
Image credits: Day 1 - Courtesy of Arkitek Studios. Used with permission. Day 2 - Courtesy of The Exploratorium. © The Exploratorium, http://www.exploratorium.edu.
Day 4 – Figure by MIT OpenCourseWare. Day 5 – Dr. Natalie Kuldell. Day 6 – Courtesy of NIH.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Expression Engineering Experiment
Lecture 1
Lecture 2
• intro to cell culture
• transfection
• intro to gene exp’n/RNAi
• luciferase
Lecture 3
Lecture 4
• off-target/nonspecific RNAi
• Writing lecture
(Neal Lerner)
Lecture 5
Lecture 6
• measuring gene express’n
• microarray analysis
(Rebecca Fry)
Lecture 7
Lecture 8
• high throughput technologies or
• review of your data
RNAi applications (no lab)
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
RNAi
Operation or Battleship?
Images removed due to copyright restrictions.
Photos of the games “Operation” and “Battleship.”
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Where were
you in 1998?
Content removed due to copyright restrictions.
News-related images:
• “France wins the World Cup”
• “Clinton under Investigation” [re: Monika Lewinski]
• President Clinton speaking at MIT commencement
• Movie poster for “Titanic”
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Source: NIH.
Image removed due to copyright restrictions.
Excerpt from Table 1: Effects of sense, antisense and mixed RNAs on progeny of
injected animals. In Fire, A., et al. “Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans.” Nature 391 (19 February 1998).
doi:10.1038/35888.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
small-RNA revolution
# of publications:
Image removed due to
copyright restrictions.
Cover of Science 298 no. 5602
(20 December 2002).
“New roles for RNAs.”
~15 in 1998
~1000 in 2000
~ 4500 in 2007 (so far)
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
RNA interference vocabulary
Transfection
siRNA
Dicer
RISC
Courtesy of Arkitek Studios. Used with permission.
Source: RNAi Interface animation for nature.com (http://www.nature.com/focus/rnai/animations/index.html)
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Nomenclature for siRNAs
+1
TT 3’-OH
5’-PO4
3’-OH
TT
5’-PO4
+19
RNA
AUG
DNA ATG
TAC
UAA
TAA
ATT
“sense”
template
“coding strand” “template strand”
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Nomenclature for siRNAs
TT
“sense”
“antisense” TT
RNA
AUG
DNA ATG
TAC
RISC
UAA
TAA
ATT
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Nomenclature for siRNAs
TT
TT
RNA
AUG
DNA ATG
TAC
RISC
UAA
TAA
ATT
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Delivery vs expression of dsRNA
Methods of delivering double-stranded RNA
Organism
Method
C. elegans
Drosophila
Trypanosomes
dsRNA
C. elegans
Drosophila
Trypanosomes
Plants
Stem-loop
expression
5'
3'
dsRNA
Trypanosomes
Dual promoter
5'
3'
Plants
3'
dsRNA
5'
Virus
Figure by MIT OpenCourseWare.
See “Post-transcriptional gene silencing by dsRNA.”
Nature Reviews Genetics 2001; 2:110-119.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
RNAi: siRNA vs miRNA
RISC
biochemistry
unites RNAi
Figure removed due to copyright restrictions.
fig from Svoboda review in Curr Opin Mol Ther. 2007 9(3):248
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
RNAi (on paper)
Highly effective
>90% reduction in gene expression
Highly specific
only targeted mRNA gets degraded
Reproducible
effects same with each expt’l repeat
Image removed due to
copyright restrictions.
Photo of the game “Operation.”
And wouldn’t it also be great if it
was genetically stable and
applicable in many cell types
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
siRNA in practice
Image removed due to
copyright restrictions.
Photo of the game “Battleship.”
Target gene expression
(% of control)
Design ambiguities
150
Firefly luciferase gene + siRNAs
120
90
60
30
0
siRNAs starting every 2 bp in gene
Courtesy of Anastasia Khvorova. Used with permission.
Reynolds, A., et al. "Rational siRNA design for RNA interference."
Nature Biotechnology 22, 326 - 330 (2004). doi:10.1038/nbt936.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
siRNA: design rules
shRNAs
5’ to 3’ unwinding:
easier end loads into
RISC
Synthetic siRNAs
or
Intracellular
processing
A. Drosha
B. Dicer
RISC
Undesired loading
A at position 3 on
sense strand
not G or C at
position 19 on sense
strand
RISC
Desired loading
Figure by MIT OpenCourseWare.
⌧ Empirically determined
⌧ Mechanism not established
⌧ Sequence and structural features of target
almost certainly influential
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
siRNA in practice: Off-target effects
Western blot
functionally
unrelated
proteins
loading
control
Some siRNAs directed against menin
decrease p53 and p21
Courtesy of National Academy of Sciences, U. S. A. Used with permission.
Source: Scacheri, P. C., et al. "Short interfering RNAs can induce unexpected and divergent changes in the levels
of untargeted proteins in mammalian cells." PNAS 101 no. 7 (February 17, 2004): 1892-1897. DOI:10.1073/pnas.0308698100.
Copyright © 2004 National Academy of Sciences, U.S.A.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
siRNA: Non-specific effects
1. siRNAs
encapsulated
in liposomes
2. Injected into
mice
3. Measured
cytokines
Courtesy of Ian MacLachlan. Used with permission.
Judge, A. D., et al. "Sequence-dependent stimulation of the mammalian innate
immune response by synthetic siRNA.“ Nature Biotechnology 23 no. 4 (April 2005):
457-462. doi:10.1038/nbt1081.
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Interpreting an siRNA experiment
+/- siRNA
Image courtesy of NOAA.
1000
900
800
700
600
500
400
300
200
100
0
no siRNA
+ siRNA
Controls?
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
Summary
RNAi biology
siRNA caveats
Photo courtesy of Colby Palmer.
• Design
Controls!!
• Off-target
Courtesy of Simone Mocellin and Maurizio
Provenzano. J Trans Med 2 (2004): 39.
doi:10.1186/1479-5876-2-39.
Image removed due to
copyright restrictions.
Photo of the game “Operation.”
• Non-specific
Image removed due to
copyright restrictions.
Photo of the game “Battleship.”
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].
the end
Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MIT
OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].